Mixed allergen compositions and methods for using the same

ABSTRACT

Mixed allergen compositions of two or more different allergens are provided. In some instances, the mixed allergen compositions include: a nut allergen; an animal allergen; and at least one of: a non-nut plant allergen; a biotic agent; and a vitamin. Also provided are methods of administering the mixed allergen compositions to a subject. The mixed allergen compositions find use in a variety of applications, including health maintenance, immune balance, gut balance, immune support, health improvement and therapeutic applications.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/228,953, filed Dec. 21, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/224,004, filed Jul. 29, 2016 (U.S. Pat. No.10,166,286, issued Jan. 1, 2019), which is a continuation-in-part ofU.S. patent application Ser. No. 15/048,609, filed Feb. 19, 2016 (U.S.Pat. No. 10,064,936, issued Sep. 4, 2018), which claims priority to U.S.Patent Application No. 62/119,014, filed Feb. 20, 2015; each of which ishereby incorporated by reference in their entirety.

BACKGROUND

Allergy is a disorder of the immune system and is characterized by theoccurrence of allergic reactions to normally harmless environmentalsubstances. Allergies are caused by allergens, which may be present in awide variety of sources, including but not limited to pollens or otherplant components, dust, molds or fungi, foods, additives, latex,transfusion reactions, animal or bird danders, insect venoms,radiocontrast medium, medications or chemicals. Common allergicreactions include eczema, hives, hay fever, asthma, and reactions tovenoms. Mild allergies like hay fever are highly prevalent in the humanpopulation and cause symptoms such as allergic conjunctivitis,itchiness, and runny nose. In some people, severe allergies toenvironmental or dietary allergens or to medication may result inlife-threatening anaphylactic reactions and potentially death, if leftuntreated. Allergic reactions can occur in three distinct patterns: a)an early phase reaction or acute response, b) late phase reactions andc) potentially chronic allergic inflammation. The early phase of theallergic reaction typically occurs within minutes, or even seconds,following a first allergen exposure, where this early phase is alsocommonly referred to as the immediate allergic reaction. In the earlystages of allergy, a hypersensitivity reaction against an allergen,encountered for the first time, causes a response in Th2 cells, whichare a subset of T cells that produce the cytokine interleukin-4 (IL-4).The Th2 cells interact with B cells (lymphocytes that produce antibodiesagainst antigens) and, coupled with the effects of IL-4, stimulate the Bcells to begin production and secretion of Immunoglobulin E (IgE).

IgE plays an important role in allergies and allergic reactions. Uponintroduction of an allergen, B cells of the respective subject producelarge amounts of IgE. The IgE elicits an immune response by binding ontoreceptors found on basophils and mast cells. When activated, these cellsrelease chemical mediators such as histamine and cytokines that causethe characteristic symptoms of allergy.

A food allergy is an adverse immune response to a food allergen, e.g., afood protein. Common food allergens are found in shellfish, peanuts,tree nuts, fish, milk, eggs, soy and fresh fruits such as strawberries,mango, banana, and apple. Immunoglobulin E (IgE)-mediated food allergiesare classified as type-I immediate hypersensitivity reactions. Theseallergic reactions have an acute onset (from seconds to one hour) andthe accompanying symptoms may include angioedema (soft tissue swellingof the eyelids, face, lips, tongue, larynx and trachea); hives; itchingof the mouth, throat, eyes, or skin; gastrointestinal symptoms such asnausea, vomiting, diarrhea, stomach cramps, or abdominal pain;rhinorrhea or nasal congestion; wheezing, shortness of breath, ordifficulty swallowing; and even anaphylaxis, a severe, whole-bodyallergic reaction that can result in death. Gupta, et al. (PEDIATRICSVol. 128 No. 1 Jul. 1, 2011 pp. e9-e17) demonstrated that 1 out of 12children under the age of 21 years of age have a doctor's diagnosis offood allergies. This epidemic has been reported to be doubling every 10years for certain nuts (CDC 2009). Moreover, there are still deaths thatoccur every year due fatal food allergies. Importantly, over $24 billionis spent per year on health care/care costs for food allergic reactions(Gupta, et al. JAMA PEDIATRICS November 2013, Vol. 167, No. 11). Thiscost is largely due to about 90,000 visits to the ER per year in theU.S. due to food induced anaphylactic symptoms.

SUMMARY

Mixed allergen compositions of two or more different allergens areprovided. In some instances, the mixed allergen compositions include: anut allergen; an animal allergen; and at least one of: a non-nut plantallergen; a biotic agent; and a vitamin. Also provided are methods ofadministering the mixed allergen compositions to a subject. The mixedallergen compositions find use in a variety of applications, includinghealth maintenance, immune balance, gut balance, immune support, healthimprovement and therapeutic applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts Th2 cell proliferation in ex vivo blood samples fromsubjects fed different food allergen mixtures (units are cells/mL). (Itis noted that with respect to each of FIGS. 1 to 6 and 9 to 10, for eachgroup of bars on the graphs the order of test compositions from left toright corresponds to the order of test compositions listed in the columnat the right of the figure from top to bottom. As such, in FIG. 1, 4-6months group, the bars from left to right are the data for each of thecompositions tested from top to bottom in the column at the right handside of the figure that lists the test compositions)

FIG. 2 depicts specific IgG4 levels in plasma from subjects feddifferent food allergen mixtures (units are mg IgG4/L).

FIG. 3 depicts specific IgE levels in plasma from subjects fed differentfood allergen mixtures (units are mg IgE/L).

FIG. 4 depicts Th2 cell proliferation in ex vivo blood samples fromsubjects fed different food allergen mixtures (units are cells/mL).

FIG. 5 depicts specific IgG4 levels in plasma from subjects feddifferent food allergen mixtures (units are mg IgG4/L).

FIG. 6 depicts specific IgE levels in plasma from subjects fed the foodallergen mixtures (units are mg IgE/L).

FIG. 7 depicts T cell proliferation in response to gluten, insulin,tetanus and bacterial flagellin in ex vivo blood samples from subjectsfed different food allergen mixtures.

FIG. 8 depicts Th2 cell proliferation in ex vivo blood samples fromsubjects fed different food allergen mixtures (units are cells/mL).

FIG. 9 depicts specific IgG4 levels in plasma from subjects feddifferent food allergen/mixtures (units are mg IgG4/L).

FIG. 10 depicts specific IgE levels in plasma from subjects feddifferent food allergen/mixtures (units are mg IgE/L).

DETAILED DESCRIPTION

Mixed allergen compositions of two or more different allergens areprovided. Mixed allergen compositions described herein comprise, e.g., 2to 20 or more (e.g., 2 to 100 or more) different allergens, andoptionally a vitamin such as a vitamin selected from vitamin D, E, B6,C, A or combinations thereof. In some instances, the mixed allergencompositions include: a nut allergen; an animal allergen; and at leastone of: a non-nut plant allergen; a biotic agent; and a vitamin. Incertain embodiments, a composition may comprise allergens selected fromthe group consisting of shellfish, peanuts, tree nuts, fish, milk, egg,legume, grain, sesame, and fruit allergen. In further embodiments, acomposition may comprise allergens or allergens mixes selected from thegroup consisting of cashew, pistachio, walnut, almond, pecan, chickenegg, cow milk, peanut, shrimp, salmon, wheat, cod, crab, soy, sesameprotein allergens, and an allergen mix comprising two or more thereof.The mixed allergen compositions find use in a variety of applications,including health maintenance, immune balance, gut balance, immunesupport, health improvement and therapeutic applications.

Also provided are methods of administering the mixed allergencompositions to a subject. Described herein are methods, e.g., oftreating an autoimmune disease or inflammatory disease in a humansubject in need thereof, which, e.g., may comprise administering acomposition comprising 1 to 20 or more (e.g., 1 to 100 more) differentprotein allergens, and optionally a vitamin, such as a vitamin selectedfrom vitamin D and vitamin C. Further described are methods, e.g., forinhibiting symptoms of one or more food allergies in a human subject,which, e.g., may comprise administering a composition comprising 2 to 20or more (e.g., 2 to 100 more) different protein allergens, andoptionally a vitamin, such as a vitamin selected from vitamin D andvitamin C. Such methods are surprisingly efficacious in treating, e.g.symptoms of allergies, e.g., even as compared to a composition having asingle protein allergen. Also provided are methods of reducing theprobability of developing one or more allergies in an unborn child of apregnant woman or a child of a nursing mother, and methods of reducingthe probability of developing one or more allergies in a child having adeveloping immune system.

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to a particular method orcomposition described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof,e.g., polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Mixed Allergen Compositions

As summarized above, aspects of the invention include mixed allergencompositions. Such compositions may have uses in a variety ofapplications including, for example, immune balance and immune support.By mixed allergen composition is meant a composition that includes twoor more different allergens, where any two given allergens are differentif they are distinct from each other, e.g., they are compounds describedby different chemical formula or compositions described by differentcomponents (e.g., constituent compounds) and/or amounts thereof. Thenumber of different allergens in a composition may vary, as desired. Incertain embodiments, the mixed allergen compositions include 2 moredifferent allergens, such as 3 or more different allergens, 4 or moredifferent allergens, 5 or more different allergens, 6 or more differentallergens, 7 or more different allergens, 8 or more different allergens,9 or more different allergens, 10 or more different allergens, 15 ormore different allergens, 20 or more different allergens, 25 or moredifferent allergens, 30 or more different allergens, 40 or moredifferent allergens, 50 or more different allergens, 75 or moredifferent allergens, 100 or more different allergens, where in someinstances the number of different allergens in a given composition is100 or less, such as 75 or less, including 50 or less, e.g., 25 or less,15 or less, including 10 or less. In certain embodiments, a compositionmay comprise 2 to 20 different protein allergens, or e.g., 2 to 100different allergens, or e.g., 2 to 1000 different allergens. In furtherembodiments, a composition may comprise 6 to 20 different proteinallergens/protein allergen compositions. In certain embodiments, acomposition may consist essentially of 2 to 20 different proteinallergens, or e.g., 2 to 100 different allergens, or e.g., 2 to 1000different allergens.

Allergens present in the composition may vary, where in some instancesthe allergen present in the composition is one that induces an allergyin a susceptible subject. Allergens include any antigen, or activederivative thereof, that elicits a specific IgE response. Antigensinclude any substance that can stimulate the production of antibodiesand can combine specifically with them. Such antigens include thosecapable of eliciting an autoimmune response. In the context ofautoimmunity, the terms antigens, protein allergens and allergens areused interchangeably to include any substance that elicits an autoimmuneresponse. Antigenic determinants or epitopes are antigenic sites on amolecule. Allergens may have little or no intrinsic toxicity bythemselves, but cause a pathological condition due to their ability toelicit an IgE-associated immune response, and, upon subsequent exposure,due to their ability to elicit IgE- and/or T cell-dependenthypersensitivity reactions. As such, an “allergen” includes anysubstance which is capable of stimulating a typical hypersensitivityreaction in atopic subjects. Allergens that may be present in a givenmixed allergen composition include any substance found in a variety ofdifferent sources, such as but not limited to: foods, drugs, perfume,plants, the environment or biological systems (e.g., prokaryotic oreukaryotic cells or viruses), as well as chemical allergens. It isappreciated that reference to allergen or an allergen composition (e.g.,such as part of a provided food product or composition) may each includea plurality of different proteins as found in the naturally occurringallergen (either raw or cooked). For example, a provided food productmay include a peanut allergen composition (which would includesubstantially all peanut proteins present in e.g., defatted peanuts,ground peanuts, etc.)

Allergens of interest include nut allergens. Nut allergens are allergensthat include one or more compounds found in nuts, e.g., dry fruits thatinclude an edible kernel or meat enclosed in a woody or leathery shell.Nut allergens of interest include, but are not limited to: peanutallergens, e.g., rAra h 1, rAra h 2, rAra h 3, rAra h 8 PR-10, rAra h 9LTP, peanut complete allergen (the phrase “complete allergen” as usedherein refers to all possible antigenic components of a given foodprotein), etc.; brazil nut allergens, e.g., rBer e 1, brazil nutcomplete allergen, etc.; hazelnut or filbert allergens, e.g., rCor a 1PR-10, rCor a 8 LTP, hazel nut complete allergen, nCor a 9, rCor a 14,etc.; walnut allergens, e.g., rJug r 1, rJug r 3 LTP, walnut completeallergen, etc.; cashew allergens; pistachio allergens, e.g., pistachiocomponent allergens, pistachio complete allergen, etc.; pecan allergens,e.g., pecan component allergens, pecan complete allergen, etc.; tree nutcomponent package allergens, such as one or more allergens from cashewnut, walnut, hazelnut, brazil nut; etc.

Allergens of interest include animal allergens. Animal allergens areallergens that include one or more compounds found in animals, includingboth vertebrates and invertebrates. Vertebrate animal allergens that maybe present in mixed allergen compositions include avian allergens, suchas egg allergens, e.g., nGal d 1 Ovomucoid, n Gal d 2 Ovalbumin, nGal d3 Conalbumin, egg white complete allergen, etc.; mammalian allergens,such as milk allergens, e.g., nBos d 4 alpha-lactalbumin, nBos d 5beta-lactoglobulin, nBos d 8 Casein, nBos d Lactoferrin, milk completeallergen, etc.; fish allergens, e.g., rCyp c 1, rGad c 1, cod completeallergen, white fish allergens, pink fish allergens, etc. Invertebrateanimal allergens that may be present in mixed allergen compositionsinclude: crustacean allergens, such as shrimp allergens, e.g., rPen a 1tropomyosin, shrimp complete allergen, etc.; insect allergens, e.g., beesting venom allergen, wasp sting venom allergen, mosquito bite allergen,etc.; and the like.

Allergens of interest include non-nut plant allergens, i.e., plantallergens that are not nut allergens. Plant allergens are allergens thatinclude one or more compounds found in plants. Plant allergens ofinterest include: wheat allergens, e.g., rTri a 19 Omega-5 Gliadin,wheat complete allergen, gliadin wheat, rTri a 14 LTP, etc.; fruitallergens, e.g., kiwi allergens, e.g., rAct d 8 PR-10, kiwi completeallergen, etc.; vegetable allergens, e.g., carrot allergens, celeryallergens etc., e.g., rApi g 1.01 PR-10, rPhl p 12, celery completeallergen, CCD MUXF3 from Bromelain, etc.; legume allergens, e.g., soyallergens, chickpea allergens, etc., e.g., rGly m 4 PR-10, soy completeallergen, nGly m 5 Beta-conglycinin, nGly m 6 Glycinin, etc.; stonefruit allergens, e.g., f419, f420, f421, f95, f242, o214 rPru p 1 PR-10,rPru p 3 LTP, stone fruit primary complete allergen, CCD MUXF3 fromBromelain, etc.; oat allergens, e.g., oat component allergens, oatcomplete allergen, etc.; seed allergens, e.g., sesame allergens, e.g.,sesame seed component allergens, sesame see complete allergen, etc.

Additional types of allergens that may be present in mixed allergencompositions include, but are not limited to: non-food animal allergens,e.g., cats or dog fur and dander, cockroach calyx, dust mite excretion,etc.; drug allergens, e.g., penicillin, sulfonamides, salicylates, localanesthetics, etc.; mold spore allergens; latex allergens; metalallergens; plant pollen allergens, e.g., grass—ryegrass, timothy-grass,weeds—ragweed, plantago, nettle, Artemisia, vulgaris, Chenopodium album,sorrel, trees—birch alder, hazel, hornbeam, aesculus, willow, poplar,platanus, tilia, olea, etc.; etc.

In certain embodiments, a composition may comprise one, two, or moreallergens each independently selected from the allergens disclosed inExamples 1 or 2 herein. For example, in certain embodiments, acomposition may comprise one, two, or more allergens selected from agroup consisting of cashew (e.g., finely ground cashew from “Wellbee's”,Spring Valley, N.Y. 10977), pistachio (e.g., pistachio flour from“nuts.com”, Cranford, N.J. 07016), walnut (e.g. roasted walnut from“Holmquist Hazelnut Orchards”, Lynden Wash. 98264), pecan (e.g., groundpecan from “King Arthur Flour”, Norwich, Vt., 05055), white fish (e.g.,pacific cod from “Seattle Seafoods”, Bellevue, Wash. 98008), pink fish(e.g., sockeye salmon from “Seattle Seafoods”, Bellevue, Wash. 98008),shrimp (e.g., white shrimp from “Seattle Seafoods”, Bellevue, Wash.98008), peanut (e.g., defatted peanuts from “Byrd Mill Company”,Ashland, Va. 23005), soy (e.g., stone ground soy from “Bob's Red Mill”,Milwaukie, Oreg. 97222), hazelnut (e.g., natural hazelnuts from“Holmquist Hazelnut Orchards”, Lynden Wash. 98264), almond (e.g.,blanched almond flour from “Honeyville” Rancho Cucamonga, Calif. 91730),milk (e.g., organic non-fat dry milk powder from “Now Foods”Bloomingdale, Ill. 60108), egg (e.g., powdered egg whites with sodiumlauryl sulphate as an anti-caking agent from “Honeyville Food Products”,Honeyville, Utah 84314), crab (e.g., crab from “Seattle Seafoods”,Bellevue, Wash. 98008), wheat (e.g., wheat flour from “Arrowhead Mills”,USA. Boulder, Colo. 80301), and sesame (Sesame flour from “Dipasa”,Brownsville, Tex. 78526), and optionally, vitamin D.

The amount of a given allergen in a mixed allergen composition may vary,as desired. In some instances, the amount of a given allergen rangesfrom about 1 to about 15,000 mg, about 5 to about 15,000 mg, about 10 toabout 10,000 mg, about 15 to about 5,000 mg, about 10 to about 100 mg,or about 15 to about 100 mg. The weight percentage of a given allergenin a mixed allergen composition may vary, ranging in some instances fromabout 0.1 to about 99.9 wt. %, about 0.1 to about 15 wt. %, about 0.1 toabout 99.9 wt. %, about 15 to about 99.9 wt. %, or about 25 to about 65wt. %. The amount of a given allergen in a mixed allergen compositionmay be recited by total mass, or by protein mass, which may vary for agiven allergen depending upon the weight percentage of protein in thatallergen.

A mixed allergen composition may be a unit dosage composition, by whichis meant that it is present in a composition that is configured to beadministered to a subject as a single dose, which single dose may or maynot be part of a dosing schedule made up of two or more unit dosagesthat are administered to a subject over a given a period of time. Whilethe mass of a given unit dosage may vary, in some instances unit dosageshave a mass ranging from 300 mg to 20 grams, such as 300 mg, 400 mg, 500mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg (1 g), 1.5 g, 2 g, 3 g, 4 g,5 g, 10 g, 15 g, 20 g, and anywhere in between. In certain embodiments,any two of the mixed allergens are present in equal parts, e.g., in a1:1 ratio, such that each allergen is present in the composition inequal weight. In such embodiments where the mixed allergen compositionincludes three different allergens, the three different allergens arepresent in a 1:1:1 ratio. For example, a mixed allergen composition mayinclude the following allergens in equal parts (e.g., 1:1:1 etc. ratio):walnut, hazelnut, shrimp, salmon, hen's egg, cow's milk, peanut, cashew,almond, and wheat (e.g., about 30 mg of protein each; about 90 mg ofeach protein; or about 300 mg of each protein). In certain embodiments,the mixed allergen composition comprises almond, cashew, walnut,hazelnut, peanut, soy, wheat, hen's egg, cow's milk, white fish, pinkfish, shrimp, and sesame allergens in equal parts with a total estimatedprotein of 400 mg and 400 IU of vitamin D3.

In some embodiments, the mixed allergen compositions include a nutallergen and an animal allergen; and at least one of: a non-nut plantallergen; a biotic agent; and a vitamin. As such, in some embodiments,the mixed allergen compositions include a nut allergen, an animalallergen and a non-nut plant allergen. In such embodiments, the mixedallergen compositions may further include a biotic agent or vitamin orboth a biotic agent and a vitamin. In some embodiments, the mixedallergen compositions include a nut allergen, an animal allergen, andfurther include a biotic agent. In such embodiments, the mixed allergencompositions may further include a vitamin. In some embodiments, themixed allergen compositions include a nut allergen, an animal allergen,and further include a vitamin. In such embodiments, the mixed allergencompositions may further include a biotic agent.

Biotic agents may vary, and include both probiotics and prebiotics. Aprobiotic is generally a live eukaryotic or a prokaryotic organism whichhas a beneficial property when given to a subject. In one aspect, theprobiotic complements the existing microflora in the subject. Hence, theprobiotic agent is a live microorganism which can confer a healthbenefit to a host subject. The probiotic agent may be a culture ofmicroorganisms or provided in a dietary supplement or may be freezedried and reconstituted prior to use. A prebiotic is an agent thatfacilitates or confers growth, maintenance and/or beneficial propertiesof or on the subject's microflora. A prebiotic may include anoligosaccharide and soluble or insoluble fiber material. Examples ofprobiotic agents include, but are not limited to, species ofLactobacillus spp., Escherichia spp., Bacillus spp., Bifidobacteriumspp., Saccharomyces spp. and Streptococcus spp. Specific probioticagents that may be present in the mixed allergen compositions include:Lactobacillus spp., such as Lactobacillus acidophilus, Lactobacilluscasei, Lactobacillus casei Shirota, Lactobacillus casei immunitass,Lactobacillus johnsonii, Lactococcus lactis, Lactobacillus plantarum,Lactobacillus reuteri, Lactobacillus rhamnosus (lactobacillus GG),Lactobacillus salivarius and Lactobacillus helvetirus. The probioticmicroorganisms may be naturally occurring, attenuated or geneticallymodified to introduce new or to alter existing traits. In oneembodiment, the probiotic has been genetically modified to introduce anallergen gene or part or fragment or portion thereof which is expressedto produce recombinant microorganisms which release or expose thesubject's immune system to the allergen or an antigenic fragmentthereof. Hence, the probiotic and allergen may be combined into a singlecomponent of the mixed allergen composition. When present, the amount ofthe biotic agent in the mixed allergen composition may vary. In someinstances, the biotic agent is present in an amount ranging from 1.5 to99.9 wt. %, such as 10 to 25 wt. %.

The mixed allergen compositions may include one or more vitamins, asdesired. Vitamins that may be present in the compositions include, butare not limited to: vitamin A, e.g., in an amount ranging from 1 to35,000 IU; vitamin C, e.g., in an amount ranging from 1 to about 1,000mg; vitamin D, e.g., in an amount ranging from 1 to 4,000 IU; vitamin E,e.g., in an amount ranging from 1 to 450 IU; vitamin K, e.g., in anamount ranging from 1 to 250 mcg; vitamin B-1 (thiamin), e.g., in amountranging from 1 to 15 mg; vitamin B-2 (riboflavin), e.g., in an amountranging from 1 to 17 mg; vitamin B-3 (niacin), e.g., in an amountranging from 1 to 200 mg; vitamin B-5 (pantothenic acid), e.g., in anamount ranging from 1 to 100 mg; vitamin B-6 (pyridoxine), e.g., in anamount ranging from 1 to 30 mg; vitamin B-9 (folic acid), e.g., in anamount ranging from 1 to 4,000 mcg; vitamin B-12 (cobalamin), e.g., inan amount ranging from 1 to 250 mcg; vitamin H (biotin), e.g., in anamount ranging from 1 to 1,000 mcg of vitamin H (biotin); etc.; andcombinations thereof.

The mixed allergen compositions of the invention may be present indifferent configurations. In some instances the mixed allergencomposition is present in a solid configuration, e.g., as a powder. Whenpresent as a powder, the dimensions of the particles making up thepowder may vary, ranging in some instances from 0.1 to 1000 microns,such as 1 to 500 microns.

Also provided are physiological acceptable compositions that include themixed allergen compositions and a physiologically acceptable deliveryvehicle. The mixed allergen compositions can be incorporated into avariety of formulations for administration to a subject. Moreparticularly, the mixed allergen compositions can be formulated intophysiological acceptable compositions by combination with appropriate,physiologically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants and aerosols and topicalcompositions. The formulations may be designed for administration via anumber of different routes, including oral, buccal, sublingual, rectal,parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc.,administration.

The physiological compositions may be in a form suitable for oral use,for example, as foods, tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsions, hard or softcapsules, or syrups or elixirs, gums, etc. Compositions intended fororal use may be prepared according to any convenient protocol for themanufacture of pharmaceutical compositions and such compositions maycontain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents and preservingagents in order to provide palatable preparations.

In certain embodiments the composition is a food product. Food productsof interest include a disclosed mixed allergen composition incombination with a food delivery vehicle. By food delivery vehicle ismeant a delivery vehicle that is a nourishing substance that is eaten,drunk, or otherwise taken into the body to sustain life, provide energy,promote growth, etc. Examples of food delivery vehicles or food productsof interest include, but are not limited to: baby or infant formula,baby food (e.g., pureed food suitable for infant or toddlerconsumption), chips, cookies, breads, spreads, creams, yogurts, liquiddrinks, chocolate containing products, candies, ice creams, cereals,coffees, pureed food products, etc.

Also of interest as oral formulations are food supplements. Where theoral formulation is provided as a food supplement, the food supplementmay further include one or more of a sweetener, a stabilizer, aflavoring or a colorant, etc. An oral formulation according to thepresent disclosure may be provided in the form of sugar-coated tabletsor lozenges, pills, gelatin capsules, or syrups. Oral formulations maybe provided as a bulk sample, e.g., a container having multiple doses inpowder form that can be measured out by a subject, or in unit dose form,e.g., a pill, pouch, single use container, and the like.

Tablets may contain the active ingredient in admixture with non-toxicphysiologically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example,magnesium stearate, stearic acid or talc. The tablets may be uncoated orthey may be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate may be employed. They mayalso be coated by the technique described in the U.S. Pat. Nos.4,256,108; 4,166,452; and U.S. Pat. No. 4,265,874 to form osmotictherapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the mixed allergen component is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, oras soft gelatin capsules wherein the mixed allergen component is mixedwith water or an oil medium, for example peanut oil, liquid paraffin, orolive oil.

Aqueous suspensions contain the mixed allergen component in admixturewith excipients suitable for the manufacture of aqueous suspensions.Such excipients may include suspending agents, for example sodiumcarboxymethyl-cellulose, methylcellulose, hydroxy-propylmethycellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

The physiologically acceptable compositions of the invention may also bein the form of oil-in-water emulsions. The oily phase may be a vegetableoil, for example olive oil or arachis oil, or a mineral oil, for exampleliquid paraffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, preservative and flavoring and coloringagents. The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The mixed allergen components can be formulated into preparations forinjection by dissolving, suspending or emulsifying them in an aqueous ornonaqueous solvent, such as vegetable or other similar oils, syntheticaliphatic acid glycerides, esters of higher aliphatic acids or propyleneglycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

The mixed allergen components can be utilized in aerosol formulation tobe administered via inhalation. The compounds of the present inventioncan be formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the mixed allergen compositions can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. The compounds of the present invention canbe administered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

Also of interest are topical compositions, e.g., where the mixedallergen composition is combined with a topical delivery vehiclecomponent. The topical delivery vehicle component of the deliverycompositions of the invention may vary, as desired, where the particularingredients of a given delivery vehicle component will depend, at leastin part, on the nature of the particular composition. Deliverycompositions of interest include liquid formulations, such as lotions(liquids containing insoluble material in the form of a suspension oremulsion, intended for external application, including spray lotions)and aqueous solutions, semi-solid formulations, such as gels (colloidsin which the disperse phase has combined with the dispersion medium toproduce a semisolid material, such as a jelly), creams (soft solids orthick liquids) and ointments (soft, unctuous preparations), and solidformulations, such as topical patches. As such, delivery vehiclecomponents of interest include, but are not limited to: emulsions of theoil-in-water (O/W) and the water in-oil (W/O) type, milk preparations,lotions, creams, ointments, gels, serum, powders, masks, packs, sprays,aerosols or sticks.

The amount of mixed allergen composition that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Forexample, a unit formulation intended for the oral administration ofhumans may contain from 0.5 mg to 5 g of mixed allergen compositioncompounded with an appropriate and convenient amount of carrier materialwhich may vary from about 0.1 to about 95 percent of the totalcomposition, or e.g. about 0.1 to about 10 percent, about 0.1 to about 4weight percent, or about 5 to about 95 percent of the total composition.It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theage, body weight, general health, sex, diet, time of administration,route of administration, rate of excretion, drug combination and theseverity of the particular disease undergoing therapy. As such, unitdosage forms for oral or rectal administration such as syrups, elixirs,and suspensions may be provided wherein each dosage unit, for example,teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may include the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier. As described above “unit dosage forms,” includephysically discrete units suitable as unitary dosages for human andanimal subjects, each unit containing a predetermined quantity of mixedallergen composition calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle.

Methods

Aspects of the invention also include methods of administering a mixedallergen composition, e.g., as described above, to a subject. Theadministration route employed in a given method may vary, e.g.,depending on the nature of the mixed allergen composition. As reviewedabove, physiologically acceptable compositions that include a mixedallergen composition may be formulated for delivery to a subject using avariety of different administration routes, such as but not limited to:oral, buccal, sublingual, rectal, parenteral, intraperitoneal,intradermal, transdermal, intracheal, etc., administration. As such,aspects of the methods may include orally, buccaly, sublingually,rectally, parenterally, intraperitonealy, intradermally, transdermally,intracheally, etc., administering a mixed allergen composition orphysiologically acceptable composition that includes the same, e.g., asdescribed above, to a subject.

The methods described herein may be employed with a variety of differenttypes of subjects, i.e., animals, where the animals are typically“mammals” or “mammalian,” where these terms are used broadly to describeorganisms which are within the class mammalia, including the orderscarnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, andrats), lagomorpha (e.g., rabbits) and primates (e.g., humans,chimpanzees, and monkeys). In embodiments of the invention, the subjectsare humans. The subject may be infant, juveniles or adults, where insome instances the subject may be pregnant adults, e.g., as described ingreater detail below. As such, in certain embodiments, the subject is apediatric subject younger than 1 year of age, whereas in otherembodiments the subject is older, e.g., 1 year old or more, 5 years oldor more, etc., and including adults. In certain embodiments thepediatric subject is 1 month to 7 years old, e.g., 1 month to 1 yearold, 6 months to 7 years old, 6 months to 1 year old, 2 years to 4 yearsold, or 3 to 7 years old. In certain embodiments, the pediatric subjectis an unborn child of a pregnant woman or a child of a nursing mother.

The dosing schedule may vary as desired, and may depend on a number ofdifferent factors, e.g., purpose of the administration, age of thesubject, condition of the subject, nature of the physiologicallyacceptable composition, etc. In certain embodiments, a mixed allergencomposition or physiologically acceptable composition that includes thesame is administered to a subject on an hourly basis, on an every fewhours basis (e.g., every 2, 3, 4, 6 hours), on a daily basis, on anevery few days basis (e.g., every 2, 3, 4, 5, or 6 days, or, e.g., 2, 3,4, 5, or 6 times a week), on a weekly basis, on a bi-weekly basis, on amonthly basis, one a bimonthly basis, on a quarter annual basis, on asemi-annual basis, on an annual basis, etc., for a treatment period oftime, which treatment period of time may also vary, where in someinstances the treatment period of time is 1 day or longer, 1 week orlonger, 2 weeks or longer, 1 month or longer, 3 months or longer, 6months or longer, 1 year or longer, 2 years or longer, 3 years orlonger, 5 years or longer, 10 years or longer, etc., up to the life ofthe subject. In certain embodiments, the composition is administereddaily for a period of time, followed by weekly, twice weekly and/orthree times a week for a period of time. In certain embodiments, thedose of the composition is varied over time, e.g., about 1 to about 10mg (by weight protein allergen) of the composition, or of each allergen,can be administered daily for about 1 week to about 30 weeks, and thenabout 1 to about 300 mg or more (by weight protein allergen) of thecomposition, or of each allergen, can be administered weekly for about 1week to about 30 weeks. In further embodiments, the dose and frequencyof the administration can be varied two, three or more times, e.g., theadministration may comprise two, three or more step-wise, increasingdose levels, wherein each dose level is about 5 to about 100% more thanthe preceding dose level by total weight protein level, wherein eachdose level is independently administered daily or weekly for about 1 toabout 30 weeks. In certain embodiments, the composition is administeredto a subject for a time or frequency to allow sufficient time for theadministration of the composition to inhibit the symptoms of anautoimmune disorder or inflammatory disorder in a subject.

In some embodiments, the methods are methods of increasing immune healthof the subject. As the methods of these embodiments are methods ofincreasing the immune health of a subject, embodiments of such methodsresult in the subject being better able to have a healthy immuneresponse to a given challenge. Immune health or immune balance may becharacterized as a state in the body where there is not an imbalance ofinflammation. For example, when IgE is decreased and/or IgG4 increasedin plasma and/or Th2 decreased (e.g., as described in greater detailbelow in the Examples section and accompanying figures). The magnitudeof immune health enhancement may vary, where in some instances themagnitude is 2-fold or greater, e.g., 5-fold or greater, including10-fold or greater, e.g., as compared to a suitable control.

In some embodiments, the methods are methods of increasing gut health ofthe subject. As the methods of these embodiments are methods ofincreasing the gut health of a subject, embodiments of such methodsresult in the subject having an enhanced maintenance of healthymicrobiota or improving the resilience of microbiota, for instance, byreducing the numbers or colonization of pathogenic bacteria or virusesand by maintaining and improving the intestinal integrity and barrierfunction. Gut health or gut balance may be characterized as a state inthe body where there is not an imbalance of inflammation. For example,when T cell proliferation is decreased (e.g., as described in greaterdetail below in the Examples section and accompanying figures). Themagnitude of gut health enhancement may vary, where in some instancesthe magnitude is 2-fold or greater, e.g., 5-fold or greater, including10-fold or greater, e.g., as compared to a suitable control.

In some embodiments, the methods are methods of enhancing wellness ormaintaining immune balance of the subject. As the methods of theseembodiments are methods of enhancing wellness of a subject, embodimentsof such methods result in the subject having a quality or state of beinghealthy in body and mind. Wellness may be characterized as a state ofthe body where there is not an imbalance of immune problems (forexample, less IgE, more IgG4, less cell inflammation (e.g., as describedin greater detail below in the Examples section and accompanyingfigures). The magnitude of wellness enhancement may vary, where in someinstances the magnitude is 2-fold or greater, e.g., 5-fold or greater,including 10-fold or greater, e.g., as compared to a suitable control.

In some embodiments, the methods are methods of at least decreasing thepotential of the subject for developing an immune mediated condition,such as an immune-mediated inflammatory disease condition. By at leastdecreasing the potential of the subject for developing an immunemediated condition is meant that the probability of the subject fordeveloping the immune mediated condition is reduced, such that the riskof the subject for developing the immune mediated condition is reduced.For example, to determine risk reduction, if 100 different individualswere administered the composition, 20% or more of the individuals wouldshow a decrease in their immune markers, e.g., IgE, as compared to thecontrol group. The magnitude of the decrease in potential may vary,where in some instances the magnitude is 2-fold or greater, e.g., 5-foldor greater, including 10-fold or greater, e.g., as compared to asuitable control. In some instances, the methods are methods ofpreventing the subject from developing an immune mediated condition. Assuch, the methods of the invention include administering a compositionof the invention to a subject that is not known to have or does not havean immune mediated condition. While the subject may not have or may notbe known to have the immune mediated condition, the subject may be onethat is suspected to be or known to be at risk of developing the immunemediated condition.

Aspects of the invention further include methods of treating a subjectfor an immune mediated condition. By treating or treatment is meant atleast an amelioration of one or more symptoms associated with thedisease condition, e.g., immune mediated condition, afflicting thesubject, where amelioration is used in a broad sense to refer to atleast a reduction in the magnitude of a parameter, e.g., symptom,associated with the pathological condition being treated, etc. As such,treatment also includes situations where the pathological condition, orat least symptoms associated therewith, are completely inhibited, e.g.,prevented from happening, or stopped, e.g., terminated, such that thesubject no longer suffers from the pathological condition, or at leastthe symptoms that characterize the pathological condition.

Treatment may also manifest in the form of a modulation of a surrogatemarker of the disease condition. For example, where the target conditionis an allergy, e.g., as described below, Th2 cell proliferation may bereduced, e.g., as determined using the assay described in the ExamplesSection, below. The magnitude of Th2 cell proliferation reduction mayvary, and in certain instances may range from 1.2× to 10×, such as 2× to4×. Where the target condition is an allergy, e.g., as described below,specific IgG4 levels may be increased, e.g., as determined using theassay described in the Examples Section, below. The magnitude of IgG4level increase may vary, and in certain instances may range from 1.2× to100×, such as 2× to 6×. Where the target condition is an allergy, e.g.,as described below, specific IgE levels may be reduced, e.g., asdetermined using the assay described in the Examples Section, below. Themagnitude of IgE level reduction may vary, and in certain instances mayrange from 1.1× to 7×, such as 2× to 6×.

As summarized above, immune mediated conditions that are the targets ofmethods of the invention include immune-mediated inflammatoryconditions, where such conditions include, but are not limited toconditions characterized by common inflammatory pathways leading toinflammation, and which may result from, or be triggered by, adysregulation of the normal immune response. Examples of immune-mediatedinflammatory conditions include, but are not limited to: allergy,autoimmune diseases, ankylosing spondylitis, psoriasis, psoriaticarthritis, Behcet's disease, arthritis, inflammatory bowel disease(IBD), cardiovascular disease, neuromuscular disease, and infectiousdisease, etc.

Also provided are methods of treating an autoimmune disease orinflammatory disease in a human subject in need thereof, which, e.g.,may comprise administering a composition comprising 1 to 20 or more(e.g., 1 to 100 more) different protein allergens, and optionally avitamin, such as a vitamin selected from vitamin D and vitamin C. Incertain embodiments, the method of treating of an autoimmune disease orinflammatory disease is prophylactic, e.g., the treating reduces theincidence of one or more autoimmune diseases or inflammatory diseases orreduces the probability of developing one or more autoimmune diseases orinflammatory diseases in a subject. In certain embodiments, the methodof treating an autoimmune disease or inflammatory disease in a subject,e.g., comprising administrating a composition comprising 1 to 20 or more(e.g., 1 to 100 more) different protein allergens, and optionally avitamin, such as a vitamin selected from vitamin D and vitamin C,results in improved immune protection against autoimmune inflammation asmeasured by the subject's Th1 proliferation to the allergen, as comparedto levels before administration.

In certain embodiments, the autoimmune disease is selected from a groupconsisting of postmyocardial infarction syndrome, postpericardiotomysyndrome, subacute bacterial endocarditis (SBE), anti-glomerularbasement membrane nephritis, interstitial cystitis, lupus nephritis,autoimmune hepatitis, primary biliary cirrhosis (PBC), primarysclerosing cholangitis, antisynthetase syndrome, alopecia areata,autoimmune angioedema, autoimmune progesterone dermatitis, autoimmuneurticarial, bullous pemphigoid, cicatricial pemphigoid, dermatitisherpetiformis, discoid lupus erythematosus, epidermolysis bullosaacquisita, erythema nodosum, gestational pemphigoid, hidradenitissuppurativa, lichen planus, lichen sclerosus, linear IgA disease (LAD),morphea, pemphigus vulgaris, pityriasis lichenoides et varioliformisacuta, Mucha-Habermann disease, psoriasis, systemic scleroderma,vitiligo, Addison's disease, autoimmune polyendocrine syndrome (APS)type 1, autoimmune polyendocrine syndrome (APS) type 2, autoimmunepolyendocrine syndrome (APS) type 3, autoimmune pancreatitis (AIP),diabetes mellitus type 1, autoimmune thyroiditis, Ord's thyroiditis,Graves' disease, autoimmune oophoritis, endometriosis, autoimmuneorchitis, Sjogren's syndrome, autoimmune enteropathy, coeliac disease,Crohn's disease, microscopic colitis, ulcerative colitis,antiphospholipid syndrome (APS, APLS), aplastic anemia, autoimmunehemolytic anemia, autoimmune lymphoproliferative syndrome, autoimmuneneutropenia, autoimmune thrombocytopenic purpura, cold agglutinindisease, essential mixed cryoglobulinemia, Evans syndrome, IgG4-relateddisease, paroxysmal nocturnal hemoglobinuria, pernicious anemia, purered cell aplasia, thrombocytopenia, adiposis dolorosa, adult-onsetStill's disease, ankylosing spondylitis, CREST syndrome, drug-inducedlupus, enthesitis-related arthritis, eosinophilic fasciitis, Feltysyndrome, juvenile arthritis, Lyme disease (chronic), mixed connectivetissue disease (MCTD), palindromic rheumatism, Parry Romberg syndrome,Parsonage-Turner syndrome, psoriatic arthritis, reactive arthritis,relapsing polychondritis, retroperitoneal fibrosis, rheumatic fever,rheumatoid arthritis, sarcoidosis, Schnitzler syndrome, systemic lupuserythematosus (SLE), undifferentiated connective tissue disease (UCTD),dermatomyositis, fibromyalgia, inclusion body myositis, myositis,myasthenia gravis, neuromyotonia, paraneoplastic cerebellardegeneration, polymyositis, acute disseminated encephalomyelitis (ADEM),acute motor axonal neuropathy, anti-N-Methyl-D-Aspartate (anti-NMDA)receptor encephalitis, balo concentric sclerosis, Bickerstaff sencephalitis, chronic inflammatory demyelinating polyneuropathy,Guillain-Barré syndrome, Hashimoto's encephalopathy, idiopathicinflammatory demyelinating diseases, Lambert-Eaton myasthenic syndrome,multiple sclerosis, pattern II, Oshtoran syndrome, pediatric autoimmuneneuropsychiatric disorder associated with Streptococcus (PANDAS),progressive inflammatory neuropathy, restless leg syndrome, stiff personsyndrome, sydenham chorea, transverse myelitis, autoimmune retinopathy,autoimmune uveitis, Cogan syndrome, Graves ophthalmopathy, intermediateuveitis, ligneous conjunctivitis, Mooren's ulcer, neuromyelitis optica,opsoclonus myoclonus syndrome, optic neuritis, scleritis, Susac'ssyndrome, sympathetic ophthalmia, Tolosa-Hunt syndrome, autoimmune innerear disease (AIED), Meniere's disease, Behcet's disease, eosinophilicgranulomatosis with polyangiitis (EGPA), giant cell arteritis,granulomatosis with polyangiitis (GPA), IgA vasculitis (IgAV),Kawasaki's disease, leukocytoclastic vasculitis, lupus vasculitis,rheumatoid vasculitis, microscopic polyangiitis (MPA), polyarteritisnodosa (PAN), polymyalgia rheumatic, urticarial vasculitis, vasculitis,chronic fatigue syndrome, complex regional pain syndrome, eosinophilicesophagitis, gastritis, interstitial lung disease, POEMS syndrome,Raynaud's phenomenon, primary immunodeficiency, and pyodermagangrenosum.

In certain embodiments, a disclosed method comprises inhibiting symptomsof one or more food allergies in a human subject, comprising orallyadministering to the subject a composition comprising 2 to 20 differentprotein allergens. In certain embodiments, administration of thecomposition significantly inhibits the symptoms of a food allergy in thesubject as compared to administering to the subject a composition thatincludes only one protein allergen. The one or more food allergies maybe a single food allergy or a multiple food allergy. The target allergymay vary widely, where in some instances the target allergy is foodallergy, drug allergy, environmental allergy, animal allergy, and insectand/or bee allergy. As such, aspects of the invention include methods ofreducing the risk of a subject for developing an allergy. In certainembodiments, the methods result in the subject having a reduced risk ofdeveloping a food allergy to a food allergen that is not present in themixed allergen composition that is administered to the subject. Forexample, the administered composition may provide to the subjectallergen protection for a nut protein in the formulation as well as anut protein not found in the formulation. In certain embodiments, thereduced risk for a first food allergen is reduced to a greater extentusing the mixed formulation than would be achieved using a formulationwith only a single allergen. For example, feeding a mixed allergenformulation as described herein can result in lower risk of allergydevelopment for an allergen (e.g., with respect to Th2/IgE/IgG4analyses, as shown below) than if the formulation only included thatsingle allergen. In some cases, this phenomenon is referred to as“synergy” (e.g., cashew synergizes with pistachio, walnut with pecan,shrimp with lobster (and other crustacean), and vice-versa). Where themethod is a method of treating the subject for the allergy, as reviewedabove the method may result in at least an amelioration of one or moresymptoms associated with the allergy, e.g., as described above. Allergysymptoms that may be ameliorated, but are not limited to: eczema,asthma, atopic dermatitis, bronchospasm, cough, rhinorrhea, angioedema,gastric hypermotility, urticaria (hives), pruritis, fatigue,bradycardia, allergic rhinitis, allergic conjunctivitis, and/orhypotension. The magnitude of the symptom reduction may vary, where insome instances the magnitude is 2-fold or greater, e.g., 5-fold orgreater, including 10-fold or greater, e.g., as compared to a suitablecontrol. In some instances, treatment of an allergy results the subjectbeing cured of the allergy, such that the subject no longer suffers fromthe allergy. In some embodiments of allergy treatment methods, themethods include administering to a subject a mixed allergen compositionthat includes a nut allergen; an animal allergen; and at least one of: anon-nut plant allergen a biotic agent; and a vitamin, such as describedabove.

Also provided are methods of significantly reducing the probability ofdeveloping one or more food allergies in a subject. In certainembodiments, a subject is an unborn child of a pregnant woman or a childof nursing mother, and the method comprises administering to thepregnant or nursing mother a disclosed composition. In certainembodiments, the subject is a child having a developing immune system.Further provided are methods for desensitizing a human to one majorallergen or to two or more major allergens comprising administering tothe human a food product comprising two or more allergens, wherein thecomposition is administered according to a schedule of treatment.

In some instances, the methods are methods of treating a subject for aneosinophilic disorder. Eosinophilic disorders are disorderscharacterized by the occurrence of eosinophils in above-normal amountsin various parts of the body. Eosinophilic disorders of interestinclude, but are not limited to: eosinophilic esophagitis (esophagus);eosinophilic gastritis (stomach); eosinophilic enteritis (smallintestine); eosinophilic colitis (large intestine); hypereosinophilicsyndrome (blood and any organ); and the like. In some instances themethods are methods of reducing the risk of a subject for developing aneosinophilic disorder. The magnitude of the risk reduction may vary,where in some instances the magnitude is 2-fold or greater, e.g., 5-foldor greater, including 10-fold or greater, e.g., as compared to asuitable control.

In some instances the methods are methods of treating a subject forinflammation, where the inflammation may be a symptom of a variety ofdifferent disease conditions. Disease conditions in which theinflammation thereof may be treated according to embodiments of theinvention include, but are not limited celiac disease, multiplesclerosis, inflammatory bowel disease, eosinophilic diseases, allergy,food allergy, etc.

Aspects of the invention further include methods of at least decreasingthe potential of a fetus or suckling infant for developing an immunemediated condition, such as an immune-mediated inflammatory diseasecondition, e.g., allergy, such as described above. By at leastdecreasing the potential of the fetus or suckling infant for developingan immune mediated condition is meant that the probability of the fetusor suckling infant for developing the immune mediated condition isreduced, such that the risk of the fetus or suckling infant fordeveloping the immune mediated condition is reduced. The magnitude ofthe decrease in potential may vary, where in some instances themagnitude is 2-fold or greater, e.g., 5-fold or greater, including10-fold or greater, e.g., as compared to a suitable control. In someinstances, the methods are methods of preventing the fetus or sucklinginfant from developing an immune mediated condition. As such, themethods of the invention include administering a composition of theinvention to a mother of a fetus or suckling infant that is not known tohave or does not have an immune mediated condition. While the fetus orsuckling infant may not have or may not be known to have the immunemediated condition, the fetus or suckling infant may be one that issuspected to be or known to be at risk of developing the immune mediatedcondition.

In certain embodiments, the mixed allergen composition orphysiologically acceptable composition that includes the same isadministered to a pregnant or nursing mother of a pediatric subject,fetus or suckling infant. For example, the mixed allergen composition orphysiologically acceptable composition that includes the same may beadministered to a mother pregnant with the fetus. In such instances, themixed allergen composition or physiologically acceptable compositionthat includes the same may be administered to the mother using anyconvenient dosing schedule, e.g., as described above, starting at anyconvenient time during the pregnancy, e.g., at the start of the secondtrimester, at the start of the third trimester, etc. Where the method isa method of reducing risk in a suckling infant, the mixed allergencomposition or physiologically acceptable composition that includes thesame may be administered to a mother that is breast feeding the infant.In such instances, the mixed allergen composition or physiologicallyacceptable composition that includes the same may be administered to thebreast feeding mother using any convenient dosing schedule, e.g., asdescribed above, starting at any convenient time during the breastfeeding, e.g., at the start of lactation, 1 week after lactationcommencement, etc.

Practice of the invention will be more fully understood from theforegoing examples, which are presented herein for illustrative purposesonly, and should not be construed as limiting the invention in any way.

EXAMPLES Example 1—Mixed Allergen Assay #1 Study Design andPopulation/Allergen Treatment

For each allergen or allergen mix listed below (the allergen groups),five subjects from each of five different age groups were fed a total of300 mg of the allergen or allergen mix daily for one year. The onlyexceptions are for Formula 1 at 3× (i.e., 3× mix) and Formula 1 at 10×(i.e., 10× mix), in which the subjects were fed 900 mg and 3,000 mg ofthe formula on a daily basis, respectively. Allergen mixes wereformulated at a 1:1 ratio. Thus, for an allergen mix with 2 allergens,the mix would include 150 mg of each. Five subjects were included ineach age group that were not fed an allergen (Not Treated, or NT group,listed last in FIGS. 1-3).

The following 18 groups were randomized equally. Children ate (daily)either 1) cashew alone, 2) cashew and pistachio, 3) walnut alone, 4)walnut and pecan, 5) a protein mix containing equal parts walnut,hazelnut, shrimp, salmon, hen's egg, cow's milk, peanut, cashew, almond,and wheat (1× mix supplement at equal protein ratios), 6) a 3× mixsupplement of the same proteins as the 1× mix, 7) a 10× mix supplementof the same proteins as the 1× mix, 8) control group that followed AAPguidelines prior to October 2015, 9) white fish alone 10) white fish andpink fish, 11) shrimp alone, 12) shrimp and crab, 13) peanut alone, 14)peanut and soy, 15) hazelnut alone, 16) almond alone, 17) cow's milk,18) hen's egg, Supplement mixes were added to breast milk or to relevantfood items eaten (i.e. apple sauce, pudding, shakes, yogurt) by thedifferent age groups, eaten freshly, and parents were told to watch anddocument the full intake of the supplement daily. The protein mix wasapproximately a teaspoon daily.

Each treatment group consisted of five children from each of thefollowing five age groups: 4 to 6 months, 7 to 12 months, 1 to 3 years,3 to 5 years, and 5 years or older. The subjects were not selected basedon observed or suspected food allergy (or other allergy) profile, andthus the cohort of subjects tested included those who may have, or havethe propensity to develop, a food allergy as well as those who do not.Children were both low- and high-risk (defined by a first degreerelative with atopic dermatitis or two first degree relatives withatopy) for developing allergies, but none had a doctor's diagnosis offood allergy.

Allergen or Allergen Mix/Protein Powders

The allergens included in the study were flours/dry powders frompeanuts, almonds, cashews, walnut, pecan, hazelnut, wheat, soy, shrimp,salmon, hen's egg (sodium lauryl sulphate was added as an anti-cakingagent), cow's milk, white fish, pink fish, sesame, and crab. The detailsof the allergens and allergen mixes were as follows:

-   -   1. Cashew: Finely ground cashew from “Wellbee's”, Spring Valley,        N.Y. 10977.    -   2. Cashew and pistachio (pis): Finely ground cashew from        “Wellbee's”, Spring Valley, N.Y. 10977. Pistachio flour from        “nuts.com”, Cranford, N.J. 07016.    -   3. Walnut: Roasted walnut from “Holmquist Hazelnut Orchards”,        Lynden Wash. 98264.    -   4. Walnut and pecan: Roasted walnut from “Holmquist Hazelnut        Orchards”, Lynden Wash. 98264. Ground pecan from “King Arthur        Flour”, Norwich, Vt., 05055.    -   5. Formula 1 at 1× (1× mix): equal parts walnut (Roasted walnut        from “Holmquist Hazelnut Orchards”, Lynden Wash. 98264),        hazelnut (Natural hazelnuts from “Holmquist Hazelnut Orchards”,        Lynden Wash. 98264), shrimp (White shrimp from “Seattle        Seafoods”, Bellevue, Wash. 98008), salmon (Sockeye salmon from        “Seattle Seafoods”, Bellevue, Wash. 98008), hen's egg (Powdered        egg whites with sodium lauryl sulphate as an anti-caking agent        from “Honeyville Food Products”, Honeyville, Utah 84314), cow's        milk (Organic non-fat dry milk powder from “Now Foods”        Bloomingdale, Ill. 60108), peanut (Defatted peanuts from “Byrd        Mill Company”, Ashland, Va. 23005), cashew (Finely ground cashew        from “Wellbee's”, Spring Valley, N.Y. 10977), almond (Blanched        almond flour from “Honeyville” Rancho Cucamonga, Calif. 91730),        and wheat (Wheat flour from “Arrowhead Mills”, USA. Boulder,        Colo. 80301) (30 mg of protein each).    -   6. Formula 1 at 3× (3× mix): equal parts walnut, hazelnut,        shrimp, salmon, hen's egg, cow's milk, peanut, cashew, almond,        and wheat as above (90 mg of each protein)    -   7. Formula 1 at 10× (10× mix): equal parts walnut, hazelnut,        shrimp, salmon, hen's egg, cow's milk, peanut, cashew, almond,        and wheat as above (300 mg of each protein)    -   8. Non-treated control (NT)    -   9. White fish: Pacific cod from “Seattle Seafoods”, Bellevue,        Wash. 98008.    -   10. White fish and pink fish: Pacific cod and sockeye salmon        from “Seattle Seafoods”, Bellevue, Wash. 98008.    -   11. Shrimp: White shrimp from “Seattle Seafoods”, Bellevue,        Wash. 98008.    -   12. Shrimp and crab: White shrimp and crab from “Seattle        Seafoods”, Bellevue, Wash. 98008.    -   13. Peanut: Defatted peanuts from “Byrd Mill Company”, Ashland,        Va. 23005.    -   14. Peanut and soy: Defatted peanuts from “Byrd Mill Company”,        Ashland, Va. 23005. Stone ground soy from “Bob's Red Mill”,        Milwaukie, Oreg. 97222.    -   15. Hazelnut: Natural hazelnuts from “Holmquist Hazelnut        Orchards”, Lynden Wash. 98264.    -   16. Almond: Blanched almond flour from “Honeyville” Rancho        Cucamonga, Calif. 91730.    -   17. Milk: Organic non-fat dry milk powder from “Now Foods”        Bloomingdale, Ill. 60108.    -   18. Egg: Powdered egg whites with sodium lauryl sulphate as an        anti-caking agent from “Honeyville Food Products”, Honeyville,        Utah 84314.

The total dose of allergens used was 300 mg for single allergens and forallergen mixes unless specified otherwise. A 3× and 10× formulationcontaining 10 allergens each containing 900 mg and 3000 mg totalallergen, respectively, were also used in the study. Allergen mixes wereformulated so that equal amounts of each allergen were present in themix, so for a 2 and 10 allergen-mix formulation with 300 mg totalallergen, the amount of each allergen was 150 mg and 30 mg,respectively. Mixing occurred prior to ingestion.

Blood Sample Collection

Peripheral blood mononuclear cells (PBMCs) and plasma were extractedfrom blood via ficoll procedure and stored in liquid nitrogen and at−80° C., respectively. Allergen-specific IgE and IgG4 were measuredusing a standard ImmunoCAP assay (Phadia, Uppsala, Sweden).

Stimulation and enumeration of Th2 cell subsets

PBMCs from subjects were labeled with carboxyfluorescein diacetatesuccinimidyl ester (CFSE) and cultured with the same food allergen orfood allergen mix that was fed to the participant at 100 μg/mL oranti-CD3/CD28 (to test for nonspecific proliferation capacity) for 7days to identify T cell subsets. At day 7, cells were washed and stainedfor surface CD4, CD25, CD127, CD45RO, CD45RA, CD40L, and CD69 andintracellular Foxp3 and IL-10 along with Live/Dead staining (Invitrogen)(see T-cell flow cytometry method below). Th2 cells were defined as thecells that proliferated in response to food allergen (CFSE10) and wereCD41 IL-4, IL-13 cells. Antigen-induced T cells were also identified byisolating CD40L and CD69 double-positive cells after antigenstimulation.

T-Cell Flow Cytometry

Cells were fixed with Lyse/Fix PhosFlow buffer (BD Biosciences). Forintracellular staining, fixed cells were permeabilized with Perm BufferIII (BD Biosciences) at 48° C. for 30 minutes, followed by staining at48° C. for 20 minutes. Flow cytometry was performed with an LSRII flowcytometer (BD Biosciences). Viable cells were identified with aLive/Dead probe (Invitrogen). Phenotypes of T cells were detected withantibodies against surface CD3 (UCHT1), CD4 (SK3), CD25 (4E3), CD127(SB199), CD45RO (UCHL1), CD45RA (HI100), CD62L (DREG-56), CCR4 (1G1),and CCR8 from BD Biosciences; CCR7, CD69, and CD40L and intracellularIL-10 (JES3-19F1), IL-4 (MP4-25D2), and IL-13 (JES10-52A2) from BDBiosciences; Helios (22F6) from BioLegend; anti-CD49b from BioLegend;anti-LAG3 from R&D Systems (Minneapolis, Minn.); and Foxp3 (150D) fromBioLegend and stained per the manufacturer-recommended protocol.

Measurement of Antibody Titers

Total and allergen-specific blood IgE and IgG4 levels were measured inall subjects in the Clinical Laboratories at Stanford Hospital andClinics using a standard ImmunoCAP assay (Phadia, Uppsala, Sweden).

Statistical Analysis

Comparisons between cohorts, and between baseline and 1 year and controlgroups were evaluated with the nonparametric Mann-Whitney test, pairedWilcoxon test, and 1-way and 2-way ANOVA (GraphPad Prism Software 5.0;GraphPad Software, La Jolla, Calif), as appropriate. A P value of lessthan 0.05 was considered statistically significant.

Compliance and Safety

Compliance was excellent with no dropouts in control arm and fifteendrop outs in ingested arms total. Of those who completed the study,there were only 90 missed doses (out of a total of 240,810) asdetermined by reviews of electronic dietary records (see Table 2).Adverse events were minimal in the active with only 21 cases of mildskin rash (21/660; 0.03%). Adverse events in the control group consistedof 3 cases of mild skin rash (3/30; 10%). 15 dropped out of the study atabout 1 year in the active arm due to non-compliance, not due to safetyissues.

TABLE 1 Summary of adverse reactions in ingestion and control groupsReaction Rates Fed Control Participants (ITT analysis) N = 660 N = 30Ingestions completed 240,810 (90 missed) n/a Reactions Total 21 (0.03%)3 (10%) Mild 21 (skin rash) 3 (skin rash) Moderate 0 0 Severe 0 0

Th2 Cell Proliferation

PBMCs from each subject in allergen groups 1 to 18 were stimulated exvivo with the allergen or allergen mix fed to the subject for 7 days andTh2 cell proliferation was analyzed (as described above). It is notedthat for Formula 1 3× and Formula 1 10× groups, the PBMCs werestimulated with 3× and 10× the amount of allergen ex vivo, respectively(i.e., 300 μg/mL and 1000 μg/mL, respectively).

As shown in FIG. 1, stimulation of PBMCs from cohorts fed singleallergens or allergen mixes had a lower degree of Th2 proliferation thancontrols (non-treated 1-year and non-treated baseline), indicatingdecreases in allergic response on exposure to allergens or allergenmixes. Additionally, the allergen mixes containing 10 allergens had thelowest degree of Th2 proliferation, significantly more than that ofcohorts containing allergen mixes of 2 allergens or single allergens,indicating that diversity of food proteins further decreases allergicresponse.

Specifically, subjects in allergen groups 1 to 4 and 9 to 18 showed asomewhat reduced Th2 proliferation in response to allergen stimulationas compared to the non-treated control (Group 8; Baseline—NT). However,Th2 cell proliferation in PBMCs from subjects in allergen groups 5 to 7(fed increasing amounts of Formula 1), who were fed formulations having10 separate allergens, as described above, had significantly reduced Th2cell proliferation as compared to both the control group (NT) as well asall other antigen groups (1 to 4 and 9 to 18). This phenomenon was seenfor all subjects in all allergen groups at all ages tested, includingfor the different doses (1×, 3×, and 10× allergen mix).

Immunoglobulin Analyses

In addition to the Th2 proliferation assay above, plasma from subjectsin allergen groups 1 to 18 were analyzed for the presence of IgG4 andIgE antibodies (using standard methods, as described above), the formerbeing an indicator of a non-allergenic or non-inflammatory (oranti-allergenic or non-inflammatory) state and the latter an indicatorof pro-allergenic or pro-inflammatory a sate.

Increased IgG4 levels indicate a protective anti-inflammatory state. Asshown in FIG. 2, when compared to baseline, samples from participantsfrom all cohorts showed an increase in IgG4, with the 10-allergen mixformula showing greater IgG4 levels than from those from cohorts fedsingle allergens or 2-allergen mixes. No significant difference in IgG4was observed between the 1×, 3×, and 10× allergen mixes containing 10allergens.

Specifically, subjects in allergenic groups 1 to 4 and 9 to 18 showedmoderately increased levels of IgG4 as compared to the baseline group(Group 8; NT). However, allergen groups 5 to 7 (fed increasing amountsof Formula 1) showed significantly increased levels of IgG4 as comparedto both the baseline group as well as all other antigen groups (1 to 4and 8 to 18). This result is consistent with the results for Th2 cellproliferation as described above. Specifically, increased levels of IgG4in the plasma and reduced Th2 cell proliferation are indicators of areduced or non-allergenic or non-inflammatory state in a subject.

Increased specific IgE is indicative of an inflammatory allergic state.As shown in FIG. 3, samples from participants from all cohorts fedallergens or allergen mixes showed decreases in IgE, with the plasma ofthose fed the 10-allergen mix (1×, 3×, and 10× formulations) having IgElevels below the assay detection level. In particular, subjects inallergenic groups 1 to 4 and 9 to 17 showed significantly higher levelsof IgE in the plasma as compared to allergen groups 5 to 7 (fedincreasing amounts of Formula 1). Indeed, IgE levels in the allergengroups 5 to 7 were below the level of detection of the assay.

These results are consistent with the results for Th2 cell proliferationand the IgG4 levels as described above. Specifically, low levels of IgEand increased levels of IgG4 in the plasma coupled with reduced Th2 cellproliferation are indicators of a reduced or non-allergenic ornon-inflammatory state in a subject.

It is clear from the results above that continual feeding of a complexmixture of food allergens to subjects (e.g., in the form of Formula 1described above) at a very young age can prophylactically protect asubject from developing an allergenic or inflammatory immune profile toa wide variety of antigens. In other words, this process can induce in asubject a non-allergenic or non-inflammatory (or anti-allergenic oranti-inflammatory) state. Of particular interest in the results shownherein is the indication that feeding antigens in a complex mixtureprovides broad spectrum protection against developingallergies/inflammation that is superior to single or even doubleallergen formulas that target a single allergen (as in allergen groups1, 3, 11, 13 and 15 to 18 above) or multiple related allergens (e.g., asin allergen groups 2, 4, 10, 12, and 14 above).

The above shows that feeding subjects complex antigen mixtures will notonly protect against the development of allergies and/or inflammation tothe allergens/antigens in the complex mixture, but also to allergens orantigens that are not present in the complex mixture. In essence,feeding complex food antigen mixtures creates a general anti-allergenicor anti-inflammatory state in a subject that broadly prevents thedevelopment of allergies or inflammatory state, even toallergens/antigens not yet fed to the subject.

Mixed Allergen Compositions (+ Probiotics and/or Vitamin)

Subjects were also fed compositions comprising allergen mixes in a 1:1blend with probiotics and/or vitamins. The details of the compositionsthat were used are shown below:

-   -   1. Formula 1 at 1× (equal parts walnut, hazelnut, shrimp,        salmon, hen's egg, cow's milk, peanut, cashew, almond, and        wheat)    -   2. Non-treated control (NT)    -   3. 1 part Formula 1 at 1× (as above) with 1 part vitamin D (400        IU; Vitamin in this instance was Poly Vi Sol brand for        children).    -   4. 1 part Formula 1 at 1× (as above) with 1 part probiotics        (Probiotic in this instance was Lactobacillus; Culturelle        brand).    -   5. 1 part Formula 1 at 1× (as above) with 1 part vitamin D (400        IU; Vitamin in this instance was Poly Vi Sol brand for children)        and 1 part probiotics (Lactobacillus; Culturelle brand).    -   6. 1 part Formula 1 at 1× (as above) after baking (15 min at        350° F.).    -   7. 1 part Formula 1 at 1× (as above) after incubation at pH=2        for 15 min.        Each composition had 300 mg each of the dietary supplement        mixture, probiotics, and/or vitamins when present. Mixtures at        pH 2 were incubated in vinegar for 40 min at room temperature        before using. Mixtures that were baked were heated for 40 min at        350° F. Control received no dietary supplement.

T-Cell Proliferation

PBMCs from each subject were stimulated ex vivo with the allergen ormixed compositions described above and Th2 cell proliferation wasanalyzed as described above. As shown in FIG. 4, PBMCs on stimulation exvivo with the allergen or allergen mix under the following conditionsshowed similar Th2 proliferation: lower pH, addition of probiotics,addition of vitamin D, or after baking.

Subject samples showed similar Th2 proliferation decreases with themixture or under different conditions with the mixture. This phenomenonwas seen for all subjects in all allergen groups and at all ages tested.

Immunoglobulin Analyses

In addition to the Th2 proliferation assay above, plasma from subjectsin allergen groups were analyzed for the presence of IgG4 and IgEantibodies (using standard methods, as described above), the formerbeing an indicator of a non-allergenic or non-inflammatory (oranti-allergenic or anti-inflammatory) state and the latter an indicatorof pro-allergenic or pro-inflammatory state.

As shown in FIGS. 5 and 6, subjects showed moderately increased levelsof IgG4 as compared to control group and significantly higher levels ofIgE in the plasma. As shown in FIG. 5, similar IgG4 levels were observedunder the following different conditions with the allergen-mix: lowerpH, addition of probiotics, addition of vitamin D, or after baking. Asshown in FIG. 6, no IgE levels were observed in response to thedifferent tested mixed allergen compositions. There were similar effectsseen with the mixture or under different conditions with the mixture,with similar IgE levels observed under the following differentconditions with the allergen-mix: lower pH, addition of probiotics,addition of vitamin D, or after baking.

T-Cell Proliferation

T cell proliferation was performed (please see methods section above)and data presented in FIG. 7. The data show anti-inflammatory propertiesto gluten (i.e., celiac agent), and insulin (i.e., diabetes agent) andbacterial flagellin (inflammatory bowel disease agent). Thisdemonstrates that the mixture could also decrease inflammatory statesinvolved in diseases like celiac, diabetes and inflammatory boweldiseases.

Example 2—Mixed Allergen Assay #2 Study Design and Population/AllergenTreatment

Infants and children were seen in a research setting from 4 weeks old to3 years of age. A single allergen or allergen mix supplement wasconsumed orally daily for one year. Subjects were fed a total of 300 mgof a single allergen or allergen mix daily for one year. The onlyexceptions are for Formula 1 at 3× (i.e., 3× mix) and Formula 1 at 10×(i.e., 10× mix), in which the subjects were fed 900 mg and 3,000 mg ofthe formula on a daily basis, respectively. Allergen mixes wereformulated at a 1:1 ratio. Thus, for an allergen mix with 2 allergens,the mix would include 150 mg of each. Thirty subjects were included thatwere not fed an allergen (control group listed).

There was similar distribution of age and sex, and all children werebreast fed for at least the first 4 months of life. There was similardistribution of high risk and low risk for food allergy (i.e. high riskare those infants born to a family with one first degree relative withfood allergy or one first degree relative with atopic dermatitis or twofirst degree relatives with atopic disease). None had a doctor'sdiagnosis of food allergy.

The subjects were not selected based on observed or suspected foodallergy (or other allergy) profile, and thus the cohort of subjectstested included those who may have, or have the propensity to develop, afood allergy as well as those who do not. The total number of subjectswas 60 including control.

TABLE 2 Demographics Age at screening Control (n = 30) Fed Allergen (n =30) 1 mo-3 yrs, median (SD) 8 months (5 mo) 9 months (6 mo) High risk*17/30 18/30 *Defined by first degree relative with atopic dermatitis ortwo first degree relatives with atopy

Allergen or Allergen Mix/Protein Powders

The allergens included in the study were flours/dry powders frompeanuts, almonds, cashews, walnut, pecan, hazelnut, wheat, soy, shrimp,salmon, hen's egg (sodium lauryl sulphate was added as an anti-cakingagent), cow's milk, white fish, pink fish, and sesame. The details ofthe allergens and allergen mixes were as follows:

-   -   1. Formula 1 at 1× (1× mix): equal parts peanuts (Defatted        peanuts from “Byrd Mill Company”, Ashland, Va. 23005), almonds        (Blanched almond flour from “Honeyville” Rancho Cucamonga,        Calif. 91730), cashews (Finely ground cashew from “Wellbee's”,        Spring Valley, N.Y. 10977), walnut (Roasted walnut from        “Holmquist Hazelnut Orchards”, Lynden Wash. 98264), pecan        (Ground pecan from “King Arthur Flour”, Norwich, Vt., 05055),        hazelnut (Natural hazelnuts from “Holmquist Hazelnut Orchards”,        Lynden Wash. 98264), wheat (Wheat flour from “Arrowhead Mills”,        Boulder, Colo. 80301), soy (Stone ground soy from “Bob's Red        Mill”, Milwaukie, Oreg. 97222), shrimp (White shrimp from        “Seattle Seafoods”, Bellevue, Wash. 98008), hen's egg (Powdered        egg whites with sodium lauryl sulphate as an anti-caking agent        from “Honeyville Food Products”, Honeyville, Utah 84314), cow's        milk (Organic non-fat dry milk powder from “Now Foods”        Bloomingdale, Ill. 60108, white fish (Pacific cod from “Seattle        Seafoods”, Bellevue, Wash. 98008), pink fish (Pacific salmon        from “Seattle Seafoods”, Bellevue, Wash. 98008), and sesame        (Sesame flour from “Dipasa”, Brownsville, Tex. 78526), (30 mg of        protein each) with or without vitamin D3 (400 IU). (i.e. Early        Adaptive Tolerance Blend)    -   2. Formula 1 at 3× (3× mix): equal parts as above (90 mg of each        protein) with or without vitamin D3 (400 IU). (i.e. Early        Adaptive Tolerance Blend)    -   3. Formula 1 at 10× (10× mix): equal parts as above (300 mg of        each protein) with or without vitamin D3 (400 IU). (i.e. Early        Adaptive Tolerance Blend)    -   4. Peanut: Defatted peanuts from “Byrd Mill Company”, Ashland,        Va. 23005.    -   5. Peanut and egg: Defatted peanuts from “Byrd Mill Company”,        Ashland, Va. 23005, Powdered egg whites with sodium lauryl        sulphate as an anti-caking agent from “Honeyville Food        Products”, Honeyville, Utah 84314.    -   6. Milk: Organic non-fat dry milk powder from “Now Foods”        Bloomingdale, Ill. 60108.    -   7. Control

The total dose of allergens used was about 300 mg total for singleallergens and for allergen mixes unless specified otherwise. A 3× and10× formulation containing allergens each containing about 900 mg and3000 mg total allergen, respectively, were also used in the study.Allergen mixes were formulated so that equal amounts of each allergenwere present in the mix. Mixing occurred prior to ingestion.

Blood Sample Collection

Peripheral blood mononuclear cells (PBMCs) and plasma were extractedfrom blood via ficoll procedure and stored in liquid nitrogen and at−80° C., respectively. Allergen-specific IgE and IgG4 were measuredusing a standard ImmunoCAP assay (Phadia, Uppsala, Sweden).

Stimulation and Enumeration of Th2 Cell Subsets

PBMCs were labeled with carboxyfluorescein diacetate succinimidyl ester(CFSE) and cultured with the same food allergen or food allergen mix at100 μg/mL or anti-CD3/CD28 (to test for nonspecific proliferationcapacity) for 7 days to identify T cell subsets. At day 7, cells werewashed and stained for surface CD4, CD25, CD127, CD45RO, CD45RA, CD40L,and CD69 and intracellular Foxp3 and IL-10 along with Live/Dead staining(Invitrogen). Th2 cells were defined as the cells that proliferated inresponse to food allergen (CFSElo) and were CD41 IL-4, IL-13 cells.Antigen-induced T cells were also identified by isolating CD40L and CD69double-positive cells after antigen stimulation.

Measurement of Antibody Titers

Total and allergen-specific blood IgE and IgG4 levels were measuredusing a standard ImmunoCAP assay (Phadia, Uppsala, Sweden).

Statistical Analysis

Comparisons between cohorts, and between baseline and 1 year and controlgroups were evaluated with the nonparametric Mann-Whitney test, pairedWilcoxon test, and 1-way ANOVA (GraphPad Prism Software 5.0; GraphPadSoftware, La Jolla, Calif.), as appropriate. A P value of less than 0.05was considered statistically significant.

Compliance and Safety

Compliance was excellent with no dropouts in control arm or fed arm. Ofthose who completed the study, there were only 3 missed doses (out of atotal of approximately 10,950) as determined by reviews of electronicdietary records (see Table 3). Adverse events were minimal in the fedarm with only 2 cases of mild skin rash (2/30; 7%). Adverse events inthe control group consisted of 5 cases of mild skin rash (5/30; 17%).

TABLE 3 Summary of adverse reactions in ingestion and control groupsReaction Rates Fed Control Participants (ITT analysis) N = 30 N = 30Ingestions completed 10,950 (3 missed doses) n/a Reactions Total 2 (7%)5 (17%) Mild 2 (skin rash) 5 (skin rash) Moderate 0 0 Severe 0 0

Th2 Cell Proliferation

PBMCs were stimulated ex vivo with the an allergen or allergen mix fedto the subject for 7 days and Th2 cell proliferation was analyzed (asdescribed in the Methods section above). It is noted that for Formula 13× and Formula 1 10×, the PBMCs were stimulated with 3× and 10× theamount of allergen ex vivo, respectively (i.e., 300 μg/mL and 1000μg/mL, respectively).

As shown in FIG. 8, PBMCs from fed subjects stimulated with singleallergens or allergen mixes had a lower degree of Th2 proliferation thanstimulated PBMCs from control subjects, indicating decreases in allergicresponse on exposure to the allergen mixes.

Immunoglobulin Analyses

In addition to the Th2 proliferation assay above, plasma from subjectswere analyzed for the presence of IgG4 and IgE specific antibodies(using standard methods, as described above), the former being anindicator of a non-allergenic or non-inflammatory (or anti-allergenic ornon-inflammatory) state and the latter an indicator of pro-allergenic orpro-inflammatory state.

Increased IgG4 levels indicate a protective anti-inflammatory state. Asshown in FIG. 9, when compared to baseline, samples from fedparticipants showed an increase in IgG4, with samples from participantsfed the allergen mix formula showing greater IgG4 levels than those fromcohorts fed single or double food compositions. No significantdifference in IgG4 was observed between samples from participants fedthe 1×, 3×, and 10× allergen mixes or with vitamin D3.

Increased specific IgE is indicative of an inflammatory allergic state.As shown in FIG. 10, samples from participants fed single food and foodmixes showed decreases in IgE, with the lowest levels seen for samplesfrom participants fed the 1×, 3×, and 10× allergen mixes.

These results are consistent with the results for Th2 cell proliferationand the IgG4 levels as described above. Specifically, low levels of IgEand increased levels of IgG4 in the plasma coupled with reduced Th2 cellproliferation are indicators of a reduced or non-allergenic ornon-inflammatory state in a subject. It is clear from the results abovethat continual feeding of a simple or complex mixture of food allergensto subjects (e.g., in the form of Formula 1) at a very young age canprophylactically protect a subject from developing an allergenic orinflammatory immune profile to a wide variety of antigens. In otherwords, this process can induce in a subject a non-allergenic ornon-inflammatory (or anti-allergenic or anti-inflammatory) state.Although a single allergen product showed evidence of effect, ofparticular interest in the results shown herein is the indication thatfeeding antigens in a complex mixture provides broad spectrum protectionagainst developing allergies/inflammation that is superior to single oreven double allergen formulas that that target a single allergen.

The above shows that feeding subjects simple or complex antigen mixtureswill not only protect against the development of allergies and/orinflammation to the allergens/antigens in the complex mixture, but alsoto allergens or antigens that are not present in the complex mixture. Inessence, feeding complex food antigen mixtures creates a generalanti-allergenic or anti-inflammatory state in a subject that broadlyprevents the development of allergies or inflammatory state, even toallergens/antigens not yet fed to the subject.

Example 3—Mixed Allergen Compositions in the Treatment of AutoimmuneDisease

The mixed allergen compositions disclosed herein will be further testedfor their ability to treat or prevent a range of autoimmune diseases orinflammatory diseases.

The mixed allergen compositions will be given to subjects via differentroutes, e.g., orally, mucosal, subcutaneously, or cutaneously. Therapywill be daily or weekly, starting at small amounts (about 1 mg or less).As appropriate, it will be possible to stay on the same amount orincrease by 5-100% each week, over time, to larger amounts (about 300 mgor higher). Blood from subjects will be collected at baseline and thenduring and after therapy. Biomarkers would be predicted to show improvedimmune balance and immune protection against autoimmune inflammation.For example, Th1 proliferation to the antigen would decrease over timeduring the exposure to the mixed allergen compositions.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

What is claimed is:
 1. A unit composition comprising a mixed allergencomposition of 9 or more different allergen flours or powders, whereinthe mixed allergen composition comprises: walnut flour; cashew flour;almond flour; peanut flour; sesame flour; soy flour; wheat flour; milkpowder; and a vertebrate animal allergen selected from the groupconsisting of salmon powder, cod powder and egg powder; wherein each ofthe walnut flour, cashew flour, almond flour, peanut flour, sesameflour, soy flour, wheat flour and milk powder are present in thecomposition in equal parts by protein weight.
 2. A mixed allergencomposition of 9 different allergen flours or powders, comprising: 30 mgto 300 mg by protein weight walnut flour; 30 mg to 300 mg by proteinweight cashew flour; 30 mg to 300 mg by protein weight almond flour; 30mg to 300 mg by protein weight peanut flour; 30 mg to 300 mg by proteinweight sesame flour; 30 mg to 300 mg by protein weight soy flour; 30 mgto 300 mg by protein weight wheat flour; 30 mg to 300 mg by proteinweight milk powder; and 30 mg to 300 mg by protein weight of avertebrate animal allergen selected from the group consisting of salmonpowder, egg powder and cod powder.
 3. A mixed allergen composition of 9different allergen flours or powders, comprising: 0.1% to 15% by proteinweight walnut flour; 0.1% to 15% by protein weight cashew flour; 0.1% to15% by protein weight almond flour; 0.1% to 15% by protein weight peanutflour; 0.1% to 15% by protein weight sesame flour; 0.1% to 15% byprotein weight soy flour; 0.1% to 15% by protein weight wheat flour;0.1% to 15% by protein weight milk powder; and 0.1% to 15% by proteinweight of a vertebrate animal allergen selected from the groupconsisting of salmon powder, egg powder and cod powder.